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19-nor-vitamin D analogs with 1,2 or 3,2 heterocyclic ring |
| 7538098 |
19-nor-vitamin D analogs with 1,2 or 3,2 heterocyclic ring
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| Patent Drawings: | |
| Inventor: |
DeLuca, et al. |
| Date Issued: |
May 26, 2009 |
| Application: |
11/697,424 |
| Filed: |
April 6, 2007 |
| Inventors: |
DeLuca; Hector F. (Deerfield, WI)
Sicinski; Rafal R. (Warsaw, PL)
Glebocka; Agnieszka (Madison, WI)
Plum; Lori A. (Arena, WI)
Clagett-Dame; Margaret (Deerfield, WI)
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| Assignee: |
Wisconsin Alumni Research Foundation (Madison, WI) |
| Primary Examiner: |
Qazi; Sabiha N. |
| Assistant Examiner: |
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| Attorney Or Agent: |
Andrus, Sceales, Starke & Sawall, LLP |
| U.S. Class: |
514/167; 552/653 |
| Field Of Search: |
514/167; 552/653 |
| International Class: |
A61K 31/59; C07D 401/00 |
| U.S Patent Documents: |
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| Foreign Patent Documents: |
WO 98/41501; WO 02/058707 |
| Other References: |
Baggiolini et al., "Stereocontrolled Total Synthesis of 1.alpha.,25-Dihydroxycholecalciferol and 1.alpha.,25-Dihydroxyergocalciferol," Journalof Organic Chemistry, 51, pp. 3098-3108, (1986). cited by other.
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Fall et al, "Vitamin D Heterocyclic Analogues. Part 1: A Stereoselective Route to CD Systems with Pyrazole Rings in their Side Chains," Tetrahedron Letters 43, pp. 1433-1436, (2002). cited by other.
Glebocka et al, "New Derivative of 1.alpha.,25-Dihydroxy-19-Norvitamin D.sub.3 with 3'-Alkoxypropylidene Moiety at C-2: Synthesis, Biological Activity and Conformational Analysis," Journal of Steroid Biochemistry & Molecular Biology, vols. 89-90,pp. 25-30, (2004). cited by other.
Granja et al, "Studies on the Opening of Dioxanone and Acetal Templates and Application to the Synthesis of 1.alpha.,25-Dihydroxyvitamin D.sub.2," Journal of Organic Chemistry, vol. 58, pp. 124-131, (1993). cited by other.
Hanessian et al, "Total Synthesis of (--)-Reserpine Using the Chiron Approach," Journal of Organic Chemistry, vol. 62, pp. 465-473, (1997). cited by other.
Inhoffen et al, "Studies in the Vitamin D Series, XXI: Hydrindane Compounds from Vitamin D.sub.3," Chemische Berichte, vol. 90, pp. 664-673, (1957). cited by other.
Lythgoe et al, "Calciferol and it Relatives. Part22. A Direct Total Synthesis of Vitamin D.sub.2 and Vitamin D.sub.3," J. Chem. Soc. Perkin Trans. 1, p. 590, (1978). cited by other.
Lythgoe, "Synthetic Approaches to Vitamin D and its Relatives," Chem. Soc. Rev. 9, p. 449, (1983). cited by other.
Minicione et al, "Improved Conversion of Vitamin D.sub.2 into the Windaus Ketone and its Regioselective Hydroxylation via Organoboranes at C.sub.26, " Synthetic Communications, vol. 19 Nos. 5-6, pp. 723-735, (1989). cited by other.
Miyamoto et al, "Synthetic Studies of Vitamin D Analogues. XIV. Synthesis and Calcium Regulating Activity of Vitamin D.sub.3 Analogues Bearing a Hydroxyalkoxy Group at the 2.beta.-Position," Chem. Pharm. Bull., vol. 41, No. 6, pp. 1111-1113, (1993).cited by other.
Nishii et al, "The Development of Vitamin D.sub.3 Analogues for the Treatment of Osteoporosis," Osteoporosis Int., Suppl. 1, pp. S190-S193, (1993). cited by other.
Okamura et al, "Vitamin D: Concerning the Relationship Between Molecular Topology and Biological Function," Proc. Nat. Acad. Sci. U.S.A., vol. 71, No. 10, pp. 4194-4197 (1974). cited by other.
Okano et al, "Regulatory Activities of 2.beta.-(3-Hydroxypropoxy)-1.alpha.,25-Dihydroxy-Vitamin D.sub.3, a Novel Synthetic Vitamin D.sub.3 Derivative, on Calcium Metabolism," Biochemical and Biophysical Research Communications, vol. 163 No. 3, pp.1444-1449, (1989). cited by other.
Ono et al, "Efficient Synthesis of 2-Modified 1.alpha.,25-Dihydroxy-19-norvitamin D.sub.3 with Julia Olefination: High Potency in Induction of Differentiation on HL-60 Cells," Journal of Orgainic Chemistry, vol. 68, pp. 7407-7415, (2003). cited byother.
Ostrem et al, "24- and 26-homo-1,25-dihydroxyvitamin D.sub.3: Preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro," Proc. Natl. Acad. Sci. USA, vol. 84, pp. 2610-2614, (1987). cited by other.
Perlman et al, "1.alpha.,25-Dihydroxy-19-Nor-Vitamin D.sub.3. A Novel Vitamin D-Related Compound with Potential Therapeutic Activity," Tetrahedron Letters, vol. 31 No. 13, pp. 1823-1824, (1990). cited by other.
Perlman et al, "Novel Synthesis of 19-Nor-Vitamin D Compounds," Tetrahedron Letters, vol. 32 No. 52, pp. 7663-7666, (1991). cited by other.
Peterson et al, "Studies of the Ketone Obtained from the Ozonolysis of Vitamin D. Molecular Mechanics Calculations for It and Related Hydrindanones," Journal of Organic Chemistry, vol. 51 No. 11, pp. 1948-1954 (1986). cited by other.
Plum et al, "Biologically Active Noncalcemic Analogs of 1.alpha.,25-Dihydroxyvitamin D with an Abbreviated Side Chain Containing No Hydroxyl," PNAS, vol. 101 No. 18, pp. 6900-6904, (2004). cited by other.
Posner et al, "Stereocontrolled Total Synthesis of Calcitriol Derivatives: 1,25-Dihydroxy-2-(4'-hydroxybutyl)vitamin D.sub.3 Analogs of an Osteoporosis Drug," Journal of Organic Chemistry, vol. 59 No. 25, pp. 7855-7861, (1994). cited by other.
Posner et al, "2-Fluoroalkyl A-Ring Analogs of 1,25-Dihydroxyvitamin D.sub.3. Stereocontrolled Total Synthesis via Intramolecular and Intermolecular Diets--Alder Cycloadditions. Preliminary Biological Testing," Journal of Organic Chemistry, vol. 60No. 14, pp. 4617-4628, (1995). cited by other.
Rochel et al, "The Crystal Structure of the Nuclear Receptor for Vitamin D Bound to Its Natural Ligand," Molecular Cell, vol. 5, pp. 173-179, (2000). cited by other.
Sardina et al, "Studies on the Synthesis of Side-Chain Hydroxylated Metabolites of Vitamin D. 2. Stereocontrolled Synthesis of 25-Hydroxyvitamin D.sub.2," J. Org. Chem., 51, pp. 1264-1269, (1986). cited by other.
Sicinski et al, "New 1.alpha.,25-Dihydroxy-19-Norvitamin D.sub.3 Compounds of High Biological Activity: Synthesis and Biological Evaluation of 2-Hydroxymethyl, 2-Methyl, and 2-Methylene Analogues," Journal of Medical Chemistry, 41, pp. 4662-4674,(1998). cited by other.
Sicinski et al, "New Highly Calcemic 1.alpha.,25-Dihydroxy-19-Norvitamin D.sub.3 Compounds with Modified Side Chain: 26,27-Dihomo- and 26,27-Dimethylene Analogs in 20S-Series," Steroids, vol. 67, pp. 247-256, (2002). cited by other.
Sicinski et al, "2-Ethyl and 2-Ethylidene Analogues of 1.alpha.,25-Dihydroxy-19-Norvitamin D.sub.3: Synthesis, Conformational Analysis, Biological Activities, and Docking to the Modeled rVDR Ligand Binding Domain," Journal of Medical Chemistry, vol.45, pp. 3366-3380, (2002). cited by other.
Tocchini-Valentini et al, "Crystal Structures of the Vitamin D Receptor Complexed to Superagonist 20-epi Ligands," Proc. Natl. Acad. Sci. USA, vol. 98 No. 10, pp. 5491-5496, (2001). cited by other.
Toh et al, "Studies on a Convergent Route to Side-Chain Analogues of Vitamin D: 25-Hydroxy-23-Oxavitamin D.sub.3," J. Org. Chem., 48, 1414, (1983). cited by other.
Windaus et al, "The Constitution of Vitamin D.sub.2 Part II," Annalen der Chemie, 524, pp. 295-299, (1936). cited by other.
Yoshida et al, "Efficient on Convergent Coupling Route for the Short-step Synthesis of Enantiopure 2.alpha.- and 2.beta.-Alkylated 1.alpha.,25-Dihydroxy-19-norvitamin D.sub.3 Analogues," Synlett, No. 8, pp. 1175-1179, (2003). cited by other.
Sicinski et a, "An Analog of 1.alpha.,25-Dihydroxy-19-Norvitamin D.sub.3 with the 1.alpha.-Hydroxy Group Fixed in the Axial Position Lacks Biological Activity in vitro," Journal of Steroid Biochemistry & Molecular Biology, vol. 103, pp. 293-297,(2007). cited by other.
PCT International Search Report, PCT/IB2007/003641, dated Jun. 10, 2008. cited by other. |
|
| Abstract: |
19-nor-vitamin D analogs having an additional heterocyclic ring connecting the 3.beta.-oxygen and carbon-2 or the 1.alpha.-oxygen and carbon-2 of the A-ring of the analog, and pharmaceutical uses therefore, are described. These compounds exhibit significant activity in mobilization of bone, making them therapeutic agents for the treatment or prophylaxis of osteoporosis, osteomalacia, osteopenia, renal osteodystrophy and hypoparathyroidism. |
| Claim: |
What is claimed is:
1. A compound having the formula: ##STR00010## where Y is selected from the group consisting of hydrogen and a hydroxy-protecting group, and where the group R represents analkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00011## where Z in the above side chain structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond inthe side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00012## where m and n, independently, represent the integers from 0 to 5, where R.sup.1 is selected fromhydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxy substituent, and where each of R.sup.2, R.sup.3, and R.sup.4,independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy or protected-hydroxy substituent, and where R.sup.1 and R.sup.2, takentogether, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 andR.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen orsulfur atom.
2. The compound of claim 1 wherein Y is hydrogen.
3. A pharmaceutical composition containing an effective amount of at least one compound as claimed in claim 1 together with a pharmaceutically acceptable excipient.
4. The pharmaceutical composition of claim 3 wherein said effective amount comprises from about 0.01 .mu.g to about 10 mg per gram of composition.
5. The pharmaceutical composition of claim 3 wherein said effective amount comprises from about 0.1 .mu.g to about 1 mg per gram of composition.
6. A method of treating a metabolic bone disease comprising administering to a subject with said disease an effective amount of a compound having the formula: ##STR00013## where Y is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where the group R represents an alkyl hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00014## where Z in the above side chain structure is selected from Y, --OY,--CH.sub.2OY, --C.dbd.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00015## where m and n, independently,represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy orprotected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy,protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or--(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
7. The method of claim 6 where the disease is senile osteoporosis.
8. The method of claim 6 where the disease is postmenopausal osteoporosis.
9. The method of claim 6 where the disease is steroid-induced osteoporosis.
10. The method of claim 6 where the disease is low bone turnover osteoporosis.
11. The method of claim 6 where the disease is osteomalacia.
12. The method of claim 6 wherein the compound is administered orally.
13. The method of claim 6 wherein the compound is administered parenterally.
14. The method of claim 6 wherein the compound is administered transdermally.
15. The method of claim 6 wherein the compound is administered rectally.
16. The method of claim 6 wherein the compound is administered nasally.
17. The method of claim 6 wherein the compound is administered sublingually.
18. The method of claim 6 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
19. The method of claim 6 wherein the compound is a 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 compounds having the formula: ##STR00016##
20. A method of treating osteopenia comprising administering to a subject with osteopenia an effective amount of a compound having the formula: ##STR00017## where Y is selected from the group consisting of hydrogen and a hydroxy-protectinggroup, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00018## where Z in the above side chain structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CYand --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00019## where m and n, independently, represent the integersfrom 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxy substituent, and whereeach of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy or protected-hydroxysubstituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is aninteger from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or C.sub.1-5 alkyl andwherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.
21. The method of claim 20 wherein the compound is administered orally.
22. The method of claim 20 wherein the compound is administered parenterally.
23. The method of claim 20 wherein the compound is administered transdermally.
24. The method of claim 20 wherein the compound is administered rectally.
25. The method of claim 20 wherein the compound is administered nasally.
26. The method of claim 20 wherein the compound is administered sublingually.
27. The method of claim 20 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
28. The method of claim 6 wherein the compound is a 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 compounds having the formula: ##STR00020##
29. A method of treating renal osteodystrophy comprising administering to a subject with renal osteodystrophy an effective amount of a compound having the formula: ##STR00021## where Y is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00022## where Z in the above side chain structure is selected from Y, --OY,--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00023## where m and n, independently,represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy orprotected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy,protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or--(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
30. The method of claim 29 wherein the compound is administered orally.
31. The method of claim 29 wherein the compound is administered parenterally.
32. The method of claim 29 wherein the compound is administered transdermally.
33. The method of claim 29 wherein the compound is administered rectally.
34. The method of claim 29 wherein the compound is administered nasally.
35. The method of claim 29 wherein the compound is administered sublingually.
36. The method of claim 29 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
37. The method of claim 29 wherein the compound is a 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 compounds having the formula: ##STR00024##
38. A compound having the formula: ##STR00025## where Y is selected from the group consisting of hydrogen and a hydroxy-protecting group, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R mayrepresent a side chain of the formula: ##STR00026## where Z in the above side chain structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Yis selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00027## where m and n, independently, represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro,trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxy substituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl,hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy or protected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene grouphaving a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group--(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogenatom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
39. The compound of claim 38 wherein Y is hydrogen.
40. A pharmaceutical composition containing an effective amount of at least one compound as claimed in claim 38 together with a pharmaceutically acceptable excipient.
41. The pharmaceutical composition of claim 40 wherein said effective amount comprises from about 0.01 .mu.g to about 10 mg per gram of composition.
42. The pharmaceutical composition of claim 40 wherein said effective amount comprises from about 0.1 .mu.g to about 1 mg per gram of composition.
43. A method of treating a metabolic bone disease comprising administering to a subject with said disease an effective amount of a compound having the formula: ##STR00028## where Y is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00029## where Z in the above side chain structure is selected from Y, --OY,--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00030## where m and n, independently,represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy orprotected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy,protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or--(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
44. The method of claim 43 where the disease is senile osteoporosis.
45. The method of claim 43 where the disease is postmenopausal osteoporosis.
46. The method of claim 43 where the disease is steroid-induced osteoporosis.
47. The method of claim 43 where the disease is low bone turnover osteoporosis.
48. The method of claim 43 wherein the disease is osteomalacia.
49. The method of claim 43 wherein the compound is administered orally.
50. The method of claim 43 wherein the compound is administered parenterally.
51. The method of claim 43 wherein the compound is administered transdermally.
52. The method of claim 43 wherein the compound is administered rectally.
53. The method of claim 43 wherein the compound is administered nasally.
54. The method of claim 43 wherein the compound is administered sublingually.
55. The method of claim 43 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
56. The method of claim 43 wherein the compound is a 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 compounds having the formula: ##STR00031##
57. A method of treating osteopenia comprising administering to a subject with osteopenia an effective amount of a compound having the formula: ##STR00032## where Y is selected from the group consisting of hydrogen and a hydroxy-protectinggroup, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00033## where Z in the above side chain structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CYand --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00034## where m and n, independently, represent the integersfrom 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxy substituent, and whereeach of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy or protected-hydroxysubstituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is aninteger from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q-- where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or C.sub.1-5 alkyl andwherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.
58. The method of claim 57 wherein the compound is administered orally.
59. The method of claim 57 wherein the compound is administered parenterally.
60. The method of claim 57 wherein the compound is administered transdermally.
61. The method of claim 57 wherein the compound is administered rectally.
62. The method of claim 57 wherein the compound is administered nasally.
63. The method of claim 57 wherein the compound is administered sublingually.
64. The method of claim 57 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
65. The method of claim 57 wherein the compound is a 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 analog having the formula: ##STR00035##
66. A method of treating renal osteodystrophy comprising administering to a subject with renal osteodystrophy an effective amount of a compound having the formula: ##STR00036## where Y is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where the group R represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain of the formula: ##STR00037## where Z in the above side chain structure is selected from Y, --OY,--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of the structure: ##STR00038## where m and n, independently,represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy orprotected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group .dbd.CR.sup.2R.sup.3, or the group--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy,protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--, --CR.sub.1R.sub.2-- or--(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
67. The method of claim 66 wherein the compound is administered orally.
68. The method of claim 66 wherein the compound is administered parenterally.
69. The method of claim 66 wherein the compound is administered transdermally.
70. The method of claim 66 wherein the compound is administered rectally.
71. The method of claim 66 wherein the compound is administered nasally.
72. The method of claim 66 wherein the compound is administered sublingually.
73. The method of claim 66 wherein the compound is administered in a dosage of from about 0.01 .mu.g/day to about 10 mg/day.
74. The method of claim 66 wherein the compound is a 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 comoounds having the formula: ##STR00039##
75. A compound having the formula: ##STR00040## where Y.sub.1 is selected from the group consisting of hydrogen and a hydroxy-protecting group.
76. The compound of claim 75 wherein Y.sub.1 is an acyl group.
77. A compound having the formula: ##STR00041## where Y.sub.3 is selected from the group consisting of hydrogen and a hydroxy-protecting group.
78. The compound of claim 77 wherein Y.sub.3 is hydrogen.
79. The compound of claim 77 wherein Y.sub.3 is t-butyldimethylsilyl.
80. A compound having the formula: ##STR00042## where Y.sub.1, Y.sub.2, and Y.sub.3, which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group.
81. The compound of claim 80 wherein Y.sub.1, Y.sub.2, and Y.sub.3 are each hydrogen. |
| Description: |
BACKGROUND OF THE INVENTION
The natural hormone, 1.alpha.,25-dihydroxyvitamin D.sub.3 and its analog in ergosterol series, i.e. 1.alpha.,25-dihydroxyvitamin D.sub.2 are known to be highly potent regulators of calcium homeostasis in animals and humans, and more recentlytheir activity in cellular differentiation has been established, Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Many structural analogs of these metabolites have been prepared and tested, including 1.alpha.-hydroxyvitamin D.sub.3,1.alpha.-hydroxyvitamin D.sub.2, various side chain homologated vitamins and fluorinated analogs. Some of these compounds exhibit an interesting separation of activities in cell differentiation and calcium regulation. This difference in activity may beuseful in the treatment of a variety of diseases as renal osteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis, and certain malignancies.
In 1990, a new class of vitamin D analogs was discovered, i.e. the so called 19-nor-vitamin D compounds, characterized by the replacement of the ring A exocyclic methylene group (carbon 19), typical of the vitamin D system, by two hydrogen atoms. Biological testing of such 19-nor-analogs (e.g., 1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3) revealed a selective activity profile with high potency in inducing cellular differentiation, with very low calcium mobilizing activity. Thus, these compoundsare potentially useful as therapeutic agents for the treatment of malignancies, or the treatment of various skin disorders. Two different methods of synthesis of such 19-nor-vitamin D analogs have been described (Perlman et al., Tetrahedron Letters 31,1823 (1990); Perlman et al., Tetrahedron Letters 32, 7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191). A few years later, analogs of 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 substituted at 2-position with hydroxy or alkoxy groups (DeLuca etal., U.S. Pat. No. 5,536,713) were synthesized. It has been established that they exhibit interesting and selective activity profiles. All these studies indicate that binding sites in vitamin D receptors can accommodate different substituents at C-2in the synthesized vitamin D analogs.
In a continuing effort to explore the 19-nor class of pharmacologically important vitamin D compounds, analogs which are characterized by the transposition of the ring A exocyclic methylene group from carbon 10 (C-10) to carbon 2 (C-2), i.e.2-methylene-19-nor-vitamin D compounds have been recently synthesized and tested (Sicinski et al., J. Med. Chem., 41, 4662 (1998); Sicinski et al., Steroids 67, 247 (2002); DeLuca et al., U.S. Pat. Nos. 5,843,928, 5,936,133 and 6,382,071). Molecularmechanics studies, performed on these analogs, showed that a change of ring-A conformation can be expected resulting in the "flattening" of the cyclohexanediol ring. From molecular mechanics calculations and NMR studies their A-ring conformationalequilibrium was established to be ca. 6:4 in favor of the conformer that has an equatorial 1.alpha.-OH. Introduction of the 2-methylene group into 19-nor-vitamin D carbon skeleton changes the character of its (1.alpha.- and 3.beta.-) A-ring hydroxyls;they are both now in the allylic positions, similar to the 1.alpha.-hydroxyl group (crucial for biological activity) in the molecule of the natural hormone, 1.alpha.,25-(OH).sub.2D.sub.3. It was found that 1.alpha.,25-dihydroxy-2-methylene-19-norvitaminD analogs are characterized by significant biological potency, enhanced dramatically in compounds with an "unnatural" (20S)-configuration.
Very recently, 2-ethylidene analogs of 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 have been synthesized. It turned out that such modification of the ring A results in significant biological potency of compounds, especially enhanced in theE-geometrical isomers, Sicinski et al., J. Med. Chem., 45, 3366 (2002). Interestingly, it has been established that E-isomers have A-ring conformational equilibrium considerably shifted to one particular chair form, that possessing 1.alpha.-hydroxyl inan equatorial orientation. Also, the analogs which are characterized by the presence of substituted propylidene moiety at C-2 have also been synthesized and preliminary biological tests indicated strong and selective (intestinal) calcemic activity ofthe E-geometrical isomers.
A-ring conformational equilibrium in vitamin D compounds has attracted considerable research interest for more than 30 years. Development of NMR spectroscopy and force field calculation methods made it possible to establish, or even predict, theproportion of equilibrating .alpha.- and .beta.-chair A-ring forms. Parallel to these studies another, closely related problem has been discussed in the literature, namely the correlation of A-ring conformation with biological activities of vitamin Dcompounds. As early as in 1974 it was proposed [Okamura et al., Proc. Natl. Acad. Sci. USA, 71, 4194 (1974)] that equatorial orientation of 1.alpha.-hydroxy group (i.e., the .beta.-chair form) is necessary for the calcium regulation ability. Recently, Moras reported the crystal structures of hVDR ligand binding domain (LBD) bound to the natural hormone [Moras et al, Moll. Cell, 5, 173 (2000)] and the ligands with unnatural configuration at C-20, [Moras et al, Proc. Natl. Acad. Sci. USA,98, 5491 (2001)] and it became clear that vitamin D receptor binds (at least in the crystalline state) to vitamin D analogs having their A-rings in .beta.-chair conformation. It seemed, therefore, interesting to synthesize a vitamin D analog that couldonly assume the opposite .alpha.-chair conformation of its ring A, and as a consequence, possesses 1.alpha.-hydroxy group in the axial orientation.
As a continuation of the search for biologically active 2-alkylidene-19-norvitamin D compounds, analogs which are characterized by the presence of an additional ring and "flattening bond" system [Corey et al, J. Org. Chem., 45, 757 (1980)] havealso been synthesized and tested. Such 19-norvitamin D compounds seemed interesting targets because structural constrains of their molecules would prevent their ring A from flipping over to the alternative .beta.-chair form, effectively "freezing" theA-ring .alpha.-chair conformation.
SUMMARY OF THE INVENTION
The present invention is directed toward 1.alpha.,25-dihydroxy and 3.beta.,25-dihydroxy-19-nor-vitamin D.sub.3 analogs, their biological activity, and various pharmaceutical uses for these compounds.
A class of 1.alpha.-hydroxylated vitamin D compounds not known heretofore are the vitamin D isomers having the A-ring exocyclic methylene moiety at C-10 removed and possessing an additional ring connecting 3.beta.-oxygen and C-2. Also, theirgeometrical isomers possessing an additional ring connecting 1.alpha.-oxygen and C-2 represent the unknown class of 19-norvitamin D compounds. Structurally these novel analogs are characterized by the general formula I and II shown below:
##STR00001##
where Y is selected from the group consisting of hydrogen and a hydroxy-protecting group, and where the group R represents any of the typical side chains known for vitamin D type compounds. Thus, R may be an alkyl, hydrogen, hydroxyalkyl orfluoroalkyl group, or R may represent a side chain of the formula:
##STR00002## where Z in the above side chain structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond in the side chain may have the cis or trans geometry, and where Y is selected from hydrogen,methyl, --COR.sup.5 and a radical of the structure:
##STR00003##
where m and n, independently, represent the integers from 0 to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be straight chain or branched and,optionally, bear a hydroxy or protected-hydroxy substituent, and where each of R.sup.2, R.sup.3, and R.sup.4, independently, is selected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain orbranched, and optionally, bear a hydroxy or protected-hydroxy substituent, and where R.sup.1 and R.sup.2, taken together, represent an oxo group, or an alkylidene group having a general formula C.sub.kH.sub.2k-- where k is an integer, the group.dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken together, represent an oxo group, or the group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where R.sup.5represents hydrogen, hydroxy, protected hydroxy, or C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20, 22, or 23 in the side chain may be replaced by a nitrogen atom, or where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22, and 23, respectively, may be replaced by an oxygen or sulfur atom.
The wavy line to the carbon 20 indicates that carbon 20 may have either the R or S configuration.
Specific important examples of side chains with natural 20R-configuration are the structures represented by formulas (a), b), (c), (d) and (e) below. i.e. the side chain as it occurs in 25-hydroxyvitamin D.sub.3 (a); vitamin D.sub.3 (b);25-hydroxyvitamin D.sub.2 (c); vitamin D.sub.2 (d); and the C-24 epimer of 25-hydroxyvitamin D.sub.2 (e).
##STR00004##
The above compounds of formulae I and II exhibit a desired, and highly advantageous, pattern of biological activity. These compounds are characterized by having significant ability to mobilize calcium from bone, as compared to1.alpha.,25-dihydroxyvitamin D.sub.3. Their preferential activity on calcium mobilizing activity allows the in vivo administration of these compounds for the treatment and prophylaxis of metabolic bone diseases. Because of their preferential calcemicactivity on bone, these compounds would be preferred therapeutic agents for the treatment and prophylaxis of metabolic bone diseases such as osteoporosis, especially low bone turnover osteoporosis, steroid induced osteoporosis, senile osteoporosis orpostmenopausal osteoporosis, as well as osteomalacia, osteopenia, renal osteodystrophy and vitamin D resistant rickets. These analogs having significant bone calcium mobilizing activity while being somewhat active on cell differentiation are alsoexpected to be useful as a therapy to treat hypoparathyroidism since they are effective to raise blood calcium levels.
The compounds of the invention of formula I and II are also useful in preventing or treating obesity, inhibiting adipocyte differentiation, inhibiting SCD-1 gene transcription, and/or reducing body fat in animal subjects. Therefore, in someembodiments, a method of preventing or treating obesity, inhibiting adipocyte differentiation, inhibiting SCD-1 gene transcription, and/or reducing body fat in an animal subject includes administering to the animal subject, an effective amount of one ormore of the compounds or a pharmaceutical composition that includes one or more of the compounds of formula I and/or II. Administration of one or more of the compounds or the pharmaceutical compositions to the subject inhibit adipocyte differentiation,inhibits gene transcription, and/or reduces body fat in the animal subject.
One or more of the compounds may be present in a composition to treat or prevent the above-noted diseases and disorders in an amount from about 0.01 .mu.g/gm to about 10 mg/gm of the composition, preferably from about 0.1 .mu.g/gm to about 1mg/gm of the composition, and may be administered topically, transdermally, orally, rectally, nasally, sublingually, or parenterally in dosages of from about 0.01 .mu.g/day to about 10 mg/day, preferably from about 0.1 .mu.g/day to about 1 mg/day.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-6 illustrate various biological activities of 1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 analog 13, hereinafter referred to as "REV-B," and 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 analog 14, hereinafter referred to as "REV-A," ascompared to the native hormone 1.alpha.,25-dihydroxyvitamin D.sub.3, hereinafter "1,25(OH).sub.2D.sub.3."
FIG. 1 is a graph illustrating the relative activity of REV-A, REV-B and 1,25(OH).sub.2D.sub.3 to compete for binding with [.sup.3H]-1,25-(OH)-.sub.2-D.sub.3 to the full-length recombinant rat vitamin D receptor;
FIG. 2 is a graph illustrating the percent HL-60 cell differentiation as a function of the concentration of REV-A, REV-B and 1,25(OH).sub.2D.sub.3;
FIG. 3 is a graph illustrating the in vitro transcription activity of 1,25(OH).sub.2D.sub.3 as compared to REV-A and REV-B; and
FIG. 4 is a bar graph illustrating the bone calcium mobilization activity of 1,25(OH).sub.2D.sub.3 as compared to REV-A and REV-B, and more specifically, it shows the serum calcium change in response to a single, intraperitoneal injection inD-deficient CD-1 mice. Statistical significance (p<0.05) compared to the vehicle group is indicated by an asterisk.
FIG. 5 is a bar graph similar to FIG. 4 illustrating the bone calcium mobilization activity of 1,25(OH).sub.2D.sub.3 as compared to REV-A and REV-B, except FIG. 5 shows the serum calcium change in response to a single, intraperitoneal injectionin D-sufficient CD-1 mice given at two different doses for each compound. Statistical significance (p<0.05) compared to the vehicle group is indicated by an asterisk.
FIG. 6 is a bar graph similar to FIGS. 4 and 5 illustrating bone calcium mobilization activity of 1,25(OH).sub.2D.sub.3 as compared to REV-B, except FIG. 6 shows the serum calcium change in response to different routes of administration inD-sufficient CD-1 mice given at different doses for each compound. Statistical significance (p<0.05) compared to the vehicle group is indicated by an asterisk.
DETAILED DESCRIPTION OF THE INVENTION
As used in the description and in the claims, the term "hydroxy-protecting group" signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups(hereinafter referred to simply as "silyl" groups), and alkoxyalkyl groups. Alkoxycarbonyl protecting groups are alkyl-O--CO-- groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term "acyl" signifies an alkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl, succinyl, glutaryl group,or an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl substituted benzoyl group. The word "alkyl" as used in the description or the claims, denotes a straight-chain or branched alkyl radical of 1 to 10 carbons, in all its isomeric forms. "Alkoxy" refers to any alkyl radical which is attached by oxygen, i.e. a group represented by "alkyl-o-." Alkoxyalkyl protecting groups are groupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term "aryl" specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.
A "protected hydroxy" group is a hydroxy group derivatised or protected by any of the above groups commonly used for the temporary or permanent protection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl or alkoxycarbonyl groups, aspreviously defined. The terms "hydroxyalkyl", "deuteroalkyl" and "fluoroalkyl" refer to an alkyl radical substituted by one or more hydroxy, deuterium or fluoro groups respectively. An "alkylidene" refers to a radical having the general formulaC.sub.kH.sub.2k-- where k is an integer.
The preparation of 19-nor-vitamin D compounds of the basic structures I and II can be accomplished by a common general method, i.e. the Julia olefination involving a coupling of an unsaturated sulfone IV, easily prepared from a bicyclicWindaus-Grundmann type ketone III, with the bicyclic ketone V:
##STR00005##
In the structures III, IV and V groups Y and R represent groups defined above whereas Ar represents phenyl, substituted phenyl (preferably phenylthiazoline group) and other aromatic groups that can be suitable for the Julia olefination process,it being also understood that any functionalities in Ar that might be sensitive, or that interfere with the condensation reaction, should be avoided. The process shown above represents an application of the convergent synthesis concept, which has beenapplied effectively for the preparation of vitamin D compounds (e.g. Kittaka et al, Synlett, 8, 1175 (2003), and J. Org. Chem., 68, 7407 (2003).
Hydrindanones of the general structure III are known, or can be prepared by known methods. Specific important examples of such known bicyclic ketones are the structures with the side chains (a), (b), (c) and (d) described above, i.e. 25-hydroxyGrundmann's ketone (e) [Baggiolini et al., J. Org. Chem, 51, 3098 (1986)]; Grundmann's ketone (f) [Inhoffen et al., Chem. Ber. 90, 664 (1957)]; 25-hydroxy Windaus ketone (g) [Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] and Windaus ketone (h)[Windaus et al., Ann., 524, 297 (1936)]:
##STR00006##
For the preparation of the required bicyclic ketones of general structure V, a new synthetic route has been developed starting from bicyclic lactone 1 that was obtained from commercial (1R,3R,4S,5R)-(-)-quinic acid as described previously[Hanessian et al., J. Org. Chem. 62, 465 (1997)]. First steps of the overall process of transformation of the starting lactone 1 into the desired A-ring synthons, is shown on the SCHEME I. Thus, one of the two secondary hydroxy groups of 1 (equatorialhydroxyl at C-3) was selectively protected as t-butyldimethylsilyl ether (TBDMS) and the other was then oxidized with Dess-Martin periodinane reagent to the 4-ketone 3. The tertiary 1-hydroxyl was acetylated and the resulted acetoxy ketone 4 subjectedto the Wittig reaction with an ylide generated from the appropriate phosphonium bromide A, prepared from 3-bromo-1-propanol, and n-butyllithium. This process afforded two isomeric olefinic compounds 5a and 5b in the ratio of ca. 5:1. The subsequentsteps of the synthesis are shown on the SCHEME II. Although many different reagents can be used for deprotection of the terminal primary hydroxy group in 5b (e.g. BuSH and MgBr.sub.2 in ethyl ether and B-chlorocatecholborane in methylene chloride), atreatment with aluminum iodide in acetonitrile provided the best yield of the desired 3'-hydroxypropylidene compound 6 that was subsequently tosylated under standard conditions. Subsequent reaction of the tosylate 7 with tetrabutylammonium fluoride gavean excellent yield of the cyclized product 8. Its reduction with sodium borohydride furnished a bicyclic triol 9. Periodate cleavage of the vicinal diol and subsequent silylation of the secondary axial hydroxyl in the formed hydroxy ketone 10 providedthe desired A-ring fragment 11. This hexahydrochromenone derivative was then subjected to modified Julia olefination. The thiazoline sulphone 12 was synthesized from the Grundmann ketone 15. The synthetic path is described on SCHEME III, and itstarted from conversion of 15 to the allylic ester 16, that was then reduced to the allylic alcohol 17. This latter compound was subjected to the three-step reaction sequence involving Mitsunobu reation, oxidation and silylation. Coupling of the ketone11 with the anion generated from 12 and lithium bis(trimethylsilyl)amide, followed by removal of the silyl protecting groups gave the expected mixture of two 19-norvitamin D analogs 13 and 14 which were purified and separated by straight- andreversed-phase HPLC. Analysis of their .sup.1H NMR spectra confirmed that ring A in these compounds, due to the presence of an exocyclic double bond being a part of additional six-membered ring, is prevented from flipping and held in the single chairconformation.
Several other 19-nor-vitamin D compounds may be synthesized by the method disclosed herein using the A-ring synthon 11 and the appropriate Windaus-Grundmann ketones having the desired side chain structure.
This invention is described by the following illustrative examples. In these examples specific products identified by Arabic numerals (e.g. 1, 2, 3, etc) refer to the specific structures so identified in the preceding description and in theSCHEME I, SCHEME II, and SCHEME III.
EXAMPLES
Chemistry. Melting points (uncorrected) were determined on a Thomas-Hoover capillary melting-point apparatus. Ultraviolet (UV) absorption spectra were recorded with a Perkin-Elmer Lambda 3B U-VIS spectrophotometer in ethanol. .sup.1H nuclearmagnetic resonance (NMR) spectra were recorded at 400 and 500 MHz with a Bruker Instruments DMX-400 and DMX-500 Avance console spectrometers in deteriochloroform. .sup.13C nuclear magnetic resonance (NMR) spectra were recorded at 125 MHz with a BrukerInstruments DMX-500 Avance console spectrometer in deuteriochloroform. Chemical shifts (.delta.) are reported downfield from internal Me.sub.4Si (.delta.0.00). Electron impact (EI) mass spectra were obtained with a Micromass AutoSpec (Beverly, Mass.)instrument. High-performance liquid chromatography (HPLC) was performed on a Waters Associates liquid chromatograph equipped with a Model 6000A solvent delivery system, a Model U6K Universal injector, and a Model 486 tunable absorbance detector. THFwas freshly distilled before use from sodium benzophenone ketyl under argon.
Example I
Preparation of 1.alpha.,25-dihydroxy- and 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 analogs 13 and 14.
Referring first to SCHEME I the starting bicyclic lactone 1 was obtained from commercial (-)-quinic acid as described previously [Hanessian et al., J. Org. Chem. 62, 465 (1997)].
(a) Protection of 3-Hydroxy Group in the Lactone 1.
(1R,3R,4S,5R)-1,4-Dihydroxy-3-[(tert-butyldimethylsilyl)oxy]-6-oxa-bicyclo- [3.2.1]octan-7-one (2). To a stirred solution of lactone 1 (1.80 g, 10.34 mmol) and imidazole (2.63 g, 38.2 mmol) in anhydrous DMF (14 mL) was added t-butyldimethylsilylchloride (1.80 g, 11.9 mmol) at 0.degree. C. The mixture was stirred at 0.degree. C. for 30 min and 1 h at room temperature, poured into water and extracted with ethyl acetate and ether. The organic layer was washed several times with water, dried(MgSO.sub.4), and evaporated to give a colorless crystalline residue which was crystallized from hexane/ethyl acetate to give 2.12 g of pure 2. The mother liquors were evaporated and purified by flash chromatography. Elution with hexane/ethyl acetate(8:2) gave additional quantity of crystalline monoether 2 (0.14 g, overall yield 76%) and some quantity of crystalline isomeric (3-OH, 4-OTBDMS) ether (0.10 g, 3%).
2: m.p. 90-94.degree. C. (from hexane); [.alpha.].sup.24.sub.D -44.degree. (c 1.00 CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.095 (6H, s, 2.times.SiCH.sub.3), 0.901 (9H, s, Si-t-Bu), ca. 2.0 (2H, br m, 2.alpha.- and 2.beta.-H),2.29 (1H, ddd, J=11.6, 6.0, 2.6 Hz, 8.beta.-H), 2.63 (1H, d, J=11.6 Hz, 8.alpha.-H), 3.89 (1H, ddd, J=10.4, 7.0, 4.5 Hz, 3.beta.-H), 3.98 (1H, t, J=4.6 Hz, 4.beta.-H), 4.88 (1H, dd, J=6.0, 4.8 Hz, 5.alpha.-H); .sup.13C NMR (125 MHz) -5.0 (Si--CH.sub.3),-4.7 (Si--CH.sub.3), 17.9 [C(CH.sub.3).sub.3], 25.6 [C(CH.sub.3).sub.3], 36.4 (C.sub.8), 40.2 (C.sub.2), 65.8 (C.sub.4), 67.0 (C.sub.3), 71.9 (C.sub.1), 76.3 (C.sub.5), 177.9 (C.dbd.O), MS (EI) m/z (relative intensity) 288(M.sup.+, 1), 231 (41), 213(21), 185 (85), 75 (100); HRMS (ESI), exact mass calcd for C.sub.13H.sub.24O.sub.5SiNa (M.sup.++Na) 311.1291, measured 311.1287; Anal. Calcd for C.sub.13H.sub.24O.sub.5Si: C, 54.14, H, 8.39. Found: C, 53.94, H, 8.36.
(b) Oxidation of 4-Hydroxy Group in the Dihydroxy Lactone 2.
(1R,3R,5R)-3-[(tert-Butyldimethylsilyl)oxy]-1-hydroxy-6-oxa-bicyclo[3.2.1]- octane-4,7-dione (3). To a stirred suspension of Dess-Martin periodinane reagent (6.60 g, 15.5 mmol) in anhydrous CH.sub.2Cl.sub.2 (100 mL) was added compound 2 (3.86 g,13.4 mmol). The mixture was stirred at room temperature for 18 h, poured into water and extracted with ethyl acetate. The organic layer was washed several times with water, dried (MgSO.sub.4), and evaporated to give an oily residue which slowlycrystallized on cooling (3.67 g, 95%). TLC indicated high purity of the obtained ketone 3 which could be used in the next step without further purification. Analytical sample was obtained by recrystallization from hexane.
3: m.p. 92-95.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.040 and 0.133 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.895 (9H, s, Si-t-Bu), 2.15 (1H, dd, J=12.4, 10.4 Hz, 2.alpha.-H), 2.42 (1H, d, J=12.5 Hz, 8.alpha.-H), 2.54 (1H,ddd, J=12.4, 9.0, 3.9 Hz, 2.beta.-H), 2.86 (1H, ddd, J=12.5, 6.7, 3.9 Hz, 8.beta.-H), 4.54 (1H, dd, J=10.4, 9.0 Hz, 3.beta.-H), 4.73 (1H, d, J=6.7 Hz, 5.alpha.-H); .sup.13C NMR (125 MHz) .delta. -5.6 (Si--CH.sub.3), -4.8 (Si--CH.sub.3), 18.2[C(CH.sub.3).sub.3], 25.6 [C(CH.sub.3).sub.3], 42.3 (C.sub.8), 43.0 (C.sub.2), 70.3 (C.sub.3), 71.8 (C.sub.1), 78.7 (C.sub.5), 177.1 (C.dbd.O), 202.4 (C.sub.4); MS (EI) m/z (relative intensity) no M.sup.+, 271 (M.sup.+-CH.sub.3, 4), 229 (92), 201 (28),157 (100); HRMS (ESI) exact mass calcd for C.sub.9H.sub.13O.sub.5Si (M.sup.+-t-Bu) 229.0532, measured 229.0539; Anal. Calcd for C.sub.13H.sub.22O.sub.5Si.times.H.sub.2O: C, 51.29, H, 7.95. Found: C, 51.09, H, 7.90.
(c) Acetylation of 1-Hydroxy Group in the Hydroxy Ketone 3.
(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsi lyl)oxy]-6-oxa-bicyclo[3.2.1]octane-4,7-dione (4). Solution of hydroxy ketone 3 (1.64 g, 5.8 mmol) in anhydrous pyridine (12 mL) and acetic anhydride (5.5 mL) was stirred for 3 h at room temperature. It was poured into water and extracted with ethyl acetate. The organic layer was washed with saturated NaHCO.sub.3, saturated CuSO.sub.4 and water, dried (MgSO.sub.4), and evaporated to give an oily residue which was dissolved in hexane/ethyl acetate(8:2) and filtered through short path of silica gel. Evaporation of solvents gave pure crystalline acetate 4 (1.51 g, 81%). Analytical sample was obtained by recrystallization from hexane/ethyl acetate.
4: m.p. 134-7.degree. C.; [.alpha.].sup.24.sub.D-78.degree. (c 1.00 CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.046 and 0.141 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.901 (9H, s, Si-t-Bu), 2.17 (3H, s, CH.sub.3CO), 2.28 (1H,dd, J=12.2, 10.4 Hz, 2.alpha.-H), 2.32 (1H, d, J=12.1 Hz, 8.beta.-H), 2.65 (1H, ddd, J=12.2, 8.8, 3.9 Hz, 2.beta.-H), 3.56 (1H, ddd, J=12.1, 6.9, 3.9 Hz, 8.beta.-H), 4.58 (1H, dd, J=10.4, 8.8 Hz, 3.beta.-H), 4.80 (1H, d, J=6.9 Hz, 5.alpha.-H); .sup.13CNMR (125 MHz) .delta. -5.8 (Si--CH.sub.3), -4.9 (Si--CH.sub.3), 18.2 [C(CH.sub.3).sub.3], 20.9 (CH.sub.3--C.dbd.O), 25.6 [C(CH.sub.3).sub.3], 38.3 (C.sub.8), 40.3 (C.sub.2), 70.4 (C.sub.3), 75.3 (C.sub.1), 78.4 (C.sub.5), 169.1 (CH.sub.3--C.dbd.O),171.5 (C.dbd.O), 201.8 (C.sub.4); MS (EI) m/z (relative intensity) 328 (M.sup.+, 6), 271 (100), 256 (38), 229 (54), 211 (53); HRMS (ESI) exact mass calcd for C.sub.11H.sub.15O.sub.6Si (M.sup.+-t-Bu) 271.0638, measured 271.0646; Anal. Calcd forC.sub.15H.sub.24O.sub.6Si: C, 54.86, H, 7.37. Found: C, 54.88, H, 7.37.
(d) Preparation of the Phosphonium Bromide A.
[3-(Methoxymethoxy)propyl]triphenylphosphonium bromide (A). To a solution of bromomethyl methyl ether (1.3 mL, 16 mmol) and N,N-diisopropylethylamine (4.5 mL, 27.7 mmol) in anhydrous CH.sub.2Cl.sub.2 (50 mL) at 0.degree. C. was added3-bromo-1-propanol (1.0 mL, 11 mmol) and the mixture was stirred at 0.degree. C. for 1 h and at room temperature for 20 h. The reaction mixture was poured into 1 N HCl (150 mL), organic phase was separated and water phase was extracted withCH.sub.2Cl.sub.2. The combined organic phases were washed with water and diluted NaHCO.sub.3, dried (MgSO.sub.4), and evaporated to give a yellowish oil. The residue was purified by flash chromatography. Elution with hexane/ethyl acetate (95:5)afforded pure oily 1-bromo-3-(methoxymethoxy)propane (1.12 g, 55%):
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.13 (2H, m, CH.sub.2--CH.sub.2--CH.sub.2), 3.37 (3H, s, O--CH.sub.3), 3.53 (2H, br t, J=6.5 Hz, Br--CH.sub.2), 3.67 (2H, br t, J=5.8 Hz, CH.sub.2--CH.sub.2--O), 4.63 (2H, s, O--CH.sub.2--O).
To a solution of 1-bromo-3-(methoxymethoxy)propane (0.46 g, 2.5 mmol) in anhydrous toluene (1.5 mL) was added triphenylphoshine (0.71 g, 2.7 mmol) under argon with stirring. The mixture was heated at 100.degree. C. for 20 h and cooled to roomtemperature. The liquid was decanted and the solid residue was grounded with spatula, filtered and washed several times with ether. After drying overnight in vacuum dessicator colorless crystals of phosphonium salt A (0.98 g, 88%) could be used in theWittig reaction without further purification.
A: m.p. 165-168.degree. C., .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.96 (2H, m, CH.sub.2--CH.sub.2--CH.sub.2), 3.31 (3H, s, O--CH.sub.3), 3.85 (2H, br t, J=5.6 Hz, CH.sub.2--CH.sub.2--O), 4.00 (2H, m, P--CH.sub.2), 4.60 (2H, s,O--CH.sub.2--O), 7.70, 7.79 and 7.86 (6H, 3H and 6H, each m, Ar--H); Anal. Calcd for C.sub.23H.sub.26O.sub.2PBr: C, 62.03, H, 5.88, Br, 17.94. Found: C, 61.87, H, 5.77, Br, 17.89.
(e) Wittig Reaction of the 4-Ketone 4 with the Ylide Generated from A.
[(E)- and (Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-6-oxa-- 4-[3'-(methoxymethoxy)propylidene]bicyclo[3.2.1]octan-7-one (5a and 5b). To the phosphonium bromide A (420 mg, 0.94 mmol) in anhydrous THF (5 mL) at 0.degree. C. wasadded dropwise n-BuLi (1.6 M in hexanes, 1.12 mL, 1.8 mmol) under argon with stirring. After 5 min another portion of A was added (420 mg, 0.94 mmol) and the solution was stirred at 0.degree. C. for 10 min and then at room temperature for 20 min. Theorange-red mixture was cooled to -78.degree. C. and siphoned in 2 equal portions (30 min interval) to a solution of keto lactone 4 (300 mg, 0.91 mmol) in anhydrous THF (8 mL). The reaction mixture was stirred at -78.degree. C. and stopped by additionof brine cont. 1% HCl (3 h after addition of the first portion of the Wittig reagent). Ethyl acetate (9 mL), benzene (6 mL), ether (3 mL), sat. NaHCO.sub.3 (3 mL), and water (3 ml) were added and the mixture was vigorously stirred at room temperaturefor 18 h. Then an organic phase was separated, washed with brine, dried (MgSO.sub.4), and evaporated. The oily residue (consisting mainly with isomeric 5a and 5b in the ratio of ca. 5:1) was separated by flash chromatography on silica. Elution withhexane/ethyl acetate (85:15) resulted in partial separation of products: 29 mg of 5b, mixture of 5a and 5b (85 mg) and pure 5a (176 mg; total yield 77%). Rechromatography of the mixed fractions resulted in almost complete separation of the products.
5a: [.alpha.].sup.24.sub.D-63.degree. (c 0.60 CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.074 (6H, s, 2.times.SiCH.sub.3), 0.914 (9H, s, Si-t-Bu), 2.13 (3H, s, OCH.sub.3), 2.00 (1H, brt, J=11.2, Hz, 2.beta.-H), 2.10 (1H, d, J=10.8Hz, 8.alpha.-H), 2.34 (1H, ddd, J=11.7, 7.0, 2.9 Hz, 2.beta.-H), 2.38 and 2.43 (1H and 1H, each m, .dbd.C--CH.sub.2), 3.31 (1H, ddd, J=10.8, 6.5, 2.9 Hz, 8, --H), 3.35 (3H, s, O--CH.sub.3), 3.54 and 3.60(1H and 1H, each m, CH.sub.2--CH.sub.2--O), 4.41(1H, t, J=8.2 Hz, 3.beta.-H), 4.60 (2H, s, O--CH.sub.2--O), 5.52 (1H, d, J=6.5 Hz, 5.alpha.-H), 5.71 (1H, br t, J=7.1 Hz, .dbd.CH); .sup.13C NMR (125 MHz) 6-5.1 (Si--CH.sub.3), -4.9 (Si--CH.sub.3), 18.1 [C(CH.sub.3).sub.3], 21.1 CH.sub.3--C.dbd.O), 25.7[C(CH.sub.3).sub.3], 27.5 (CH.sub.2--CH.sub.2--C.dbd.), 40.5 (C.sub.8), 41.5 (C.sub.2), 55.2 (O--CH.sub.3), 66.7 (O--CH.sub.2--CH.sub.2), 66.8 (C.sub.3), 77.1 (C.sub.1), 73.9 (C.sub.5), 96.3 (O--CH.sub.2--O), 121.9 (.dbd.C--CH.sub.2), 136.8 (C.sub.4),169.1 (CH.sub.3--C.dbd.O), 172.9 (C.dbd.O); MS (EI) m/z (relative intensity), no M.sup.+, 383 (M.sup.+-OCH.sub.3, 3), 357 (10), 325 (44), 297 (12), 267 (15), 265 (40), 237 (89), 75 (100); HRMS (ESI) exact mass calcd for C.sub.20H.sub.34O.sub.7SiNa(M.sup.++Na) 437.1972, measured 437.1975.
5b: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.108 and 0.125 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.912 (9H, s, Si-t-Bu), 2.13 (3H, s, OCH.sub.3), 2.15 (1H, dd, J=12.6, 8.3 Hz, 2.alpha.-H), 2.31 (1H, d, J=10.8 Hz, 8.alpha.-H), 2.33 (1H,2.beta.-H overlapped with 8.alpha.-H), 2.67 and 2.73 (1H and 1H, each m, .dbd.C--CH.sub.2), 3.25 (1H, ddd, J=10.8, 6.3, 2.8 Hz, 8.beta.-H), 3.36 (3H, s, O--CH.sub.3), 3.55 (2H, m, CH.sub.2--CH.sub.2--O), 4.61 (2H, s, O--CH.sub.2--O), 4.71 (1H, br t,J.about.7 Hz, 3.beta.-H), 4.94 (1H, d, J=6.3 Hz, 5.alpha.-H), 5.64 (1H, dt, J=1.7, 7.1 Hz, .dbd.CH); .sup.13C NMR (125 MHz) .delta. -4.6 (Si--CH.sub.3), -4.5 (Si--CH.sub.3), 17.9 [C(CH.sub.3).sub.3], 21.1 (CH.sub.3--C.dbd.O), 25.7 [C(CH.sub.3).sub.3],27.8 (CH.sub.2--CH.sub.2--C.dbd.), 38.9 (C.sub.8), 41.2 (C.sub.2), 55.3 (O--CH.sub.3), 67.1 (O--CH.sub.2--CH.sub.2), 67.2 (C.sub.3), 77.1 (C.sub.1), 81.8 (C.sub.5), 96.4 (O--CH.sub.2--O), 128.9 (.dbd.C--CH.sub.2), 134.8 (C.sub.4), 169.1(CH.sub.3--C.dbd.O), 173.0 (C.dbd.O); MS (EI) m/z (relative intensity), no M.sup.+, 383 (M.sup.+-OCH.sub.3, 2), 357 (2), 325 (22), 297 (17), 267 (35), 265 (14), 237 (96), 75 (100); HRMS (ESI) exact mass calcd for C.sub.20H.sub.34O.sub.7SiNa (M.sup.++Na)437.1972, measured 437.1974.
(4Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-4-[3'-hydroxypr- opylidene]-6-oxabicyclo[3.2.1]octan-7-one (6). To a solution of 5b (26 mg, 63 .mu.mol) in anhydrous CH.sub.3CN (0.6 mL) was added AlI.sub.3 (170 mg, 0.42 mmol) at0.degree. C. under argon. The mixture was stirred at 0.degree. C. for 50 min, poured into aq Na.sub.2S.sub.2O.sub.3, and extracted with ethyl acetate. The extract was washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The residue waspurified by flash chromatography. Elution with hexane/ethyl acetate (6:4) afforded compound 6 as a colorless oil (16.5 mg, 71%).
6: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.132 and 0.144 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.925 (1H, s, C(CH.sub.3).sub.3), 2.13 (3H, s, COCH.sub.3), 2.14 (1H, dd, J=12.4, 8.8 Hz, 2.alpha.-H), 2.28 (1H, d, J=10.8 Hz, 8.alpha.-H),2.33 (1H, ddd, J=12.4, 7.0, 2.8 Hz, 2.beta.-H), 2.45 and 2.79 (2H, 2.times.m, CH--CH.sub.2), 3.29 (1H, ddd, J=10.8, 6.2, 2.8 Hz, 8.beta.-H), 3.60 and 3.71(2H, 2.times.m, CH.sub.2OH), 4.71(1H, t, J=7.8 Hz, 3.beta.-H), 4.97 (1H, d, J=6.3 Hz, 5.alpha.-H),5.65 (1H, dt, J=1.8, 7.8 Hz, .dbd.CH); .sup.13C NMR (125 MHz) .delta. -4.6 (SiCH.sub.3), 17.9 [C(CH.sub.3).sub.3], 25.8 [C(CH.sub.3).sub.3], 21.1 (COCH.sub.3), 30.1 (CH--CH.sub.2), 39.8 (C.sub.8), 41.4 (C.sub.2), 61.4 (CH.sub.2OH), 67.6 (C.sub.3), 77.0(C.sub.1), 81.2 (C.sub.5-), 128 (CH--CH.sub.2), 135.8 (C.sub.4), 169.2 (COCH.sub.3), 173.0 (CO); HRMS (ESI) exact mass calcd for C.sub.18H.sub.30O.sub.6SiNa (M.sup.++Na) 393.1709, measured 393.1690.
(4Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-6-oxa-4-[3'-(p-- toluenesulfonyloxy)propylidene]bicyclo[3.2.1]octan-7-one (7). To a solution of hydroxy compound (16 mg, 43 .mu.mol) in anhydrous pyridine (140 .mu.L) was added at0.degree. C. p-toluenesulfonyl chloride (24 mg, 127 .mu.mol) and a catalytic quantity of 4-(dimethylamino)pyridine. The mixture was stirred at 0.degree. C. for 1 h and at 6.degree. C. for 18 h. It was then poured into ice/saturated NaHCO.sub.3,shaken for 15 min and extracted with ethyl acetate and benzene. The combined extracts were washed with saturated NaHCO.sub.3, water, saturated CuSO.sub.4, water again, dried (Na.sub.2SO.sub.4) and evaporated. The residue (ca. 16 mg) was dissolved inbenzene/hexane, applied on a silica Sep-Pak cartridge, and washed with hexane/ethyl acetate (95:5, 10 mL) to remove apolar impurities. Elution with washed with hexane/ethyl acetate (85:15, 20 mL) provided a pure oily tosylate 7 (19 mg, 84%).
7: [.alpha.].sup.24.sub.D-48.degree. (c 0.80 CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.043 and 0.088 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.871 (1H, s, C(CH.sub.3).sub.3), 2.13 (3H, s, COCH.sub.3), 2.08 (1H, dd, J=12.0, 9.3Hz, 2.alpha.-H), 2.16 (1H, d, J=10.8 Hz, 8.alpha.-H), 2.28 (1H, ddd, J=12.0, 6.9, 3.0 Hz, 2.beta.-H), 2.46 (3H, s, CH.sub.3--Ar), 2.67 and 2.88 (2H, 2.times.m, CH--CH.sub.2), 3.26 (1H, ddd, J=10.8, 6.2, 3.0 Hz, 8-.beta.H), 4.05 (2H, t, J=6.4 Hz,CH.sub.2OS), 4.62 (1H, t, J.about.8 Hz, 3.beta.-H), 4.85 (1H, d, J=6.4 Hz, 5.alpha.-H), 5.43 (1H, dt, J=2, 7.5 Hz, .dbd.CH), 7.36 and 7.78 (2H and 2H, each d, J=8.3 Hz, Ar--H); HRMS (ESI) exact mass calcd for C.sub.25H.sub.36O.sub.8SSiNa (M.sup.++Na)547.1798, measured 547.1812
(1R,7R,9S)-(9-Acetoxy-6,11-dioxa-tricyclo[7.2.1.0*2,7*]dodec-2-en-10-one (8). Solution of tosylate 7 (18.8 mg, 36 .mu.mol) in dry THF (8 mL) was treated with tetrabutylammonium fluoride (1.0 M in THF, 180 .mu.l, 180 .mu.mol). The mixture wasstirred under argon at room temperature for 18 h, poured into brine, and extracted with ethyl acetate and benzene. Organic extracts were washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The oily residue was dissolved in hexane and applied ona silica Sep-Pak cartridge, and washed with hexane/ethyl acetate (85:15, 20 mL) to give a pure tricyclic product 8 (7.6 mg, 89%) as an oil.
8: [.alpha.].sup.24.sub.D -51.degree. (c 0.40 CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.02 (1H, dm, J.about.17 Hz, 4.beta.-H), 2.07 (1H, dd, J=11.6, 10.6 Hz, 8.alpha.-H), 2.11 (1H, d, J=11.0 Hz, 12.alpha.-H), 2.13 (3H, s,COCH.sub.3), 2.39 (1H, m, w/2=38 Hz, 4.alpha.-H), 2.50 (1H, ddd, J=11.6, 7.8, 3.1 Hz, 8.beta.-H), 3.35 (1H, ddd, J=11.0, 6.2, 3.1 Hz, 12.beta.-H), 3.66 (1H, dt, J=3.8, 11.4 Hz, 5.beta.-H), 4.04 (1H, dd, J=11.5, 6.3 Hz, 5.alpha.-H), 4.31 (1H, m, w/2=22Hz, 7.beta.-H), 5.00 (1H, d, J=6.2 Hz, 1.alpha.-H), 5.86 (1H, m, w/2=11 Hz, 3-H); .sup.13C NMR (125 MHz) .delta. 21.1 (COCH.sub.3), 24.5 (C.sub.4), 37.6 (C.sub.8), 40.9 (C.sub.12), 64.2 (C.sub.5), 69.1 (C.sub.7), 78.5 (C.sub.1), 77.1 (C.sub.9), 121.9(C.sub.3-), 134.6 (C.sub.2-), 169.2 (COCH.sub.3), 172.5 (C.sub.10); HRMS (ESI) exact mass calcd for C.sub.12H.sub.14O.sub.5 (M.sup.+) 238.0841, measured 238.0851
(5R,7R,8aR)-7-Hydroxymethyl-3,5,6,7,8,8a-hexahydro-2H-chromene-5,7-diol (9). To a solution of tricyclic compound 8 (9.5 mg, 40 .mu.mol) at 0.degree. C. was added NaBH.sub.4. The resultant mixture was then stirred at room temperature for 18 h,a small volume of brine/saturated NH.sub.4Cl was added, and the solvents were removed in vacuum. The residue was washed several times with warm ethanol. The ethanol extracts were combined and evaporated to dryness with benzene. The solid residue wasthen washed few times with warm chloroform. The combined chloroform extracts were concentrated to a small volume and applied on a silica Sep-Pak cartridge. Elution with hexane/ethyl acetate (1:9, 20 mL) yielded a pure semisolid triol 9 (6 mg, 75%).
9: [.alpha.].sup.24.sub.D+20.degree. (c 0.30, MeOH); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.54 (1H, t, J=12.1 Hz, 8.alpha.-H), 1.65 (1H, dd, J=14.3, 3.9 Hz, 6.alpha.-H), 1.98 (1H, dm, J.about.16 Hz, 3.beta.-H), 2.18 (1H, dt, J=14.3, 2.3Hz, 6.beta.-H), 2.29 (1H, ddd, J=12.4, 5.5, 2.3 Hz, 8.beta.-H), 2.34 (1H, br m, 3.alpha.-H), 3.60 (1H, ddd, J=11.2, 10.2, 3.8 Hz, 2.beta.-H), 3.72 and 3.81 (1H and 1H, each d, J=11.3 Hz, CH.sub.2OH), 3.94 (1H, ddd, J=11.2, 5.7, 2.0 Hz, 2.alpha.-H), 4.37(2H, m, 5.alpha.- and 8a.beta.-H), 5.84 (1H, m, w/2=11 Hz, 4-H);
.sup.13C NMR (125 MHz) .delta. 25.5 (C.sub.3), 41.3 and 41.8 (C.sub.6 and C.sub.8), 62.9 (C.sub.2), 69.2 (CH.sub.2OH), 69.4 (C.sub.8a), 72.2 (C.sub.5), 76.5 (C.sub.7), 122.8 (C.sub.4), 140.0 (C.sub.4a); HRMS (ESI) exact mass calcd forC.sub.10H.sub.14O.sub.3 (M.sup.+-H.sub.2O) 182.0943, measured 182.0949.
(5R,8aR)-5-Hydroxy-2,3,5,6,8,8a-hexahydro-chromen-7-one (10): Sodium periodate-saturated water (50 .mu.L) was added to a solution of the triol 21 (5 mg, 2.6 .mu.mol) in methanol (200 .mu.L) at 0.degree. C. The mixture was stirred at 0.degree. C. for 1 h, then thioanisole was added and stirring was continued for 10 min. The mixture was diluted with benzene/ethyl acetate (1:1, 1 mL) and filtered through a silica Sep-Pak. Then Sep-Pak was washed with additional 5 mL of the same solvent system,the combined solutions were evaporated, the residue redissolved in hexane/ethyl acetate (7:3) and applied on a silica gel Sep-Pak. Elution with the same solvent system (10 mL) provided aromatic compounds and a pure(5R,8aR)-5-hydroxy-2,3,5,6,8,8a-hexahydro-chromen-7-one (4.1 mg, 98%) was eluted with hexane/ethyl acetate (1:1, 10 mL) as a colorless oil.
10: [.alpha.].sup.24.sub.D+6.degree. (c 0.22, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 2.09 (1H, dm, J=17.6 Hz, 3.alpha.-H), 2.38 (1H, m, w/2=35 Hz, 3.beta.-H), 2.49 (1H, dd, J=13.8, 11.1 Hz, 8.alpha.-H), 2.60 (1H, dd, J=15.0, 3.7Hz, 6.alpha.-H), 2.65 (1H, dd, J=15.0, 2.5 Hz, 6.beta.-H), 2.89 (1H, ddd, J=13.8, 6.4, 1.6 Hz, 8.beta.-H), 3.68 (1H, ddd, J=11.3, 9.3, 3.9 Hz, 2.beta.-H), 3.96 (1H, ddd, J=11.3, 5.4, 2.9 Hz, 2.alpha.-H), 4.62 (1H, t, J.about.3 Hz, 5.alpha.-H), 4.67 (1H,m, w/2=24 Hz, 8a.beta.-H), 6.01 (1H, m, w/2=10 Hz, 4-H); .sup.13C NMR (125 MHz) .delta. 25.1 (C.sub.3), 47.9 (C.sub.8), 48.8 (C.sub.6), 62.5 (C.sub.2), 69.9 (C.sub.8a), 72.5 (C.sub.5), 123.3 (C.sub.4), 138.2 (C.sub.4a), 206.5 (C.sub.7); HRMS (ESI) exactmass calcd for C.sub.9H.sub.12O.sub.3Na (M.sup.++Na) 191.0684, measured 191.0676.
(5R,8aR)-5-[tert-Butyldimethylsilyl)oxy]-2,3,5,6,8,8a-hexahydro-chromen-7-- one (11). To a solution of hydroxy ketone (4 mg, 24 .mu.mol) in anhydrous methylene chloride (90 .mu.l) was added at -50.degree. C. 2,6-lutidine (7 .mu.L, 60 mmol) andtert-butyldimethylsilyl trifluoromethanesulfonate (12 .mu.L, 51 mmol). The reaction mixture was stirred at -50.degree. C. for 50 min, diluted with cold methylene chloride and poured into water. The organic phase was washed with saturated CuSO.sub.4and water, dried (Na.sub.2SO.sub.4) and evaporated. The residue was redissolved in hexane and applied on a silica gel Sep-Pak. Elution with hexane/ethyl acetate (95:5, 10 mL) provided less polar compound (2.1 mg) being TBDMS derivative of the enolether derived from 11. The desired protected hydroxy ketone 11 (3.3 mg, 49%) was eluted with hexane/ethyl acetate (9:1, 10 mL) as a colorless oil. Further elution with the same solvent system afforded 2,3,8,8a-tetrahydro-chromen-7-one (0.6 mg).
11: [.alpha.].sup.24.sub.D-9.degree. (c 0.11, CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.035 and 0.058 (3H and 3H, each s, 2.times.SiCH.sub.3), 0.838 [1H, s, C(CH.sub.3).sub.3], 2.08 (1H, dm, J=17.3 Hz, 3.alpha.-H), 2.33 (1H, m,w/2=33 Hz, 3.beta.-H), 2.46 (1H, dd, J=14.0, 10.9 Hz, 8.alpha.-H), 2.53 (2H, narr m, 6.alpha.- and 6.beta.-H), 2.86 (1H, br dd, J=14.0, 6.5 Hz, 8.beta.-H), 3.65 (1H, ddd, J=11.4, 9.0, 3.9 Hz, 2.beta.-H), 3.92 (1H, ddd, J=11.4, 4.9, 3.7 Hz, 2.alpha.-H),4.51 (1H, t, J.about.3 Hz, 5.alpha.-H), 4.61 (1H, m, w/2=22 Hz, 8a.beta.-H), 5.87 (1H, m, w/2=10 Hz, 4-H); HRMS (ESI) exact mass calcd for C.sub.15H.sub.26O.sub.3SiNa (M.sup.++Na) 305.1549, measured 305.1534.
[(1R,3aS,7aR)-7a-Methyl-1-[(R)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl]- -octahydro-inden-(4E)-ylidene]acetic acid ethyl ester (16): To a suspension of NaH (49 mg, 2.04 mmol) in anhydrous THF (1.2 mL) was added (EtO).sub.2P(O)CH.sub.2COOEt(500 ml, 2.53 mmol) at 0.degree. C. The mixture was stirred at room temperature for 10 min and lithium chloride (13 mg, 0.30 mmol) was then added. The stirring was continued for 1 h, cooled to 0.degree. C. and a solution of the protected hydroxyketone 15 (100 mg, 0.25 mmol) in THF (0.6 mL) was added. After stirring at room temperature for 70 h the reaction mixture was diluted with ethyl acetate and poured into saturated ammonium chloride. Organic phase was separated, washed with brine, dried(Na.sub.2SO.sub.4) and evaporated. The residue was separated by flash chromatography. Elution with hexane/ethyl acetate (99:1) afforded pure oily [(1R,3aS,7aR)-7a-methyl-1-[(R)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl-]-octahydro-inden-(4E)-ylidene]acetic acid ethyl ester 16 (61 mg, 52%, 65% based on recovered starting material). Further elution with hexane/ethyl acetate (97:3) gave unchanged substrate 15 (20 mg).
16: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.562 (6H, q, J=7.9 Hz, 3.times.SiCH.sub.2), 0.581 (3H, s, 7a-H.sub.3), ca. 0.94 (3H, overlapped, CH.sub.3--CH), 0.944 (9H, t, J=7.9 Hz, 3.times.SiCH.sub.2CH.sub.3), 1.187 [6H, s,C(CH.sub.3).sub.2], 1.284 (3H, t, J=7.1 Hz, CH.sub.3CH.sub.2O), 3.84 (1H, m, 5.beta.-H), 4.15 (2H, m, CH.sub.3CH.sub.2O), 5.45 (1H, br s, .dbd.CH).
2-[(1R,3 aS,7aR)-7a-Methyl-[(R)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl- ]-octahydro-inden-(4E)-ylidene]ethanol (17). Diisobutylaluminum hydride (1 M in toluene, 200 .mu.L, 0.2 mmol) was added to a stirred solution of allylic ester (29 mg, 62.mu.mol) in anhydrous toluene (0.5 mL) at -78.degree. C. under argon. The mixture was stirred at -78.degree. C. for 1 h, and the reaction was quenched by addition of potassium sodium tartrate (2 N, 1 mL), aq HCl (2 N, 1 mL), and water (12 mL). Themixture was poured into brine and extracted with ethyl acetate and ether. The combined extracts were washed with diluted NaHCO.sub.3 and brine, dried (Na.sub.2SO.sub.4) and evaporated. The residue was redissolved in hexane and applied on a silica gelSep-Pak. Elution with hexane/ethyl acetate (95:5, 20 mL, and 9:1, 10 mL) gave allylic alcohol 17 (23 mg, 87%) as a colorless oil.
17: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.563 (6H, q, J=7.9 Hz, 3.times.SiCH.sub.2), 0.554 (3H, s, 7a-H.sub.3), 0.928 (3H, d, J=7 Hz, CH.sub.3--CH), 0.945 (9H, t, J=7.9 Hz, 3.times.SiCH.sub.2CH.sub.3), 1.188 [6H, s, C(CH.sub.3).sub.2],2.63 (1H, dd, J=12.0, 4.5 Hz, 5.beta.-H), 4.20 (2H, m; after D.sub.2O d, J=7.0 Hz, CH.sub.2OH), 5.22 (1H, t, J=7.0 Hz, .dbd.CH).
(1R,3aS,7aR)-4-[2-(Benzothiazole-2-sulfonyl)-(4E)-ethylidene]-7a-methyl-1-- [(R)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl]-octahydro-indene (12). To a solution of 2-mercaptobenzotriazole (12.5 mg, 75 .mu.mol) and Ph.sub.3P (19.5 mg, 75.mu.mol) in dry methylene chloride (150 .mu.L) at 0.degree. C. was added a solution of allylic alcohol 17 (21 mg, 50 .mu.moL) in methylene chloride (150 .mu.L) followed by DIAD (14 .mu.L, 50 .mu.mol). The mixture was stirred at 0 oC for 1 h and thesolvents were evaporated in vacuo. The residue was dissolved in ethanol (300 mL), cooled to 0.degree. C. and 30% H.sub.2O.sub.2 (30 .mu.L) was added, followed by ammonium (NH.sub.4).sub.6MoO.sub.7O.sub.24.times.4H.sub.2O (12.3 mg, 10% mol). Themixture was stirred at room temperature for 3 h, poured into cold saturated Na.sub.2SO.sub.3 and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The residue was dissolved in small volumeof benzene/hexane (1:1) and applied on a silica Sep-Pak. Elution with hexane/ethyl acetate (9:1, 20 mL and 85:15, 20 mL) and removal of the solvents gave an oily product (33 mg) that was dissolved in anhydrous DMF (300 .mu.L). Imidazole (18 mg, 0.26mmol) was added followed by triethylsilyl chloride (50 .mu.L, 0.29 mmol) and the mixture was stirred at room temperature for 3 h. Ethyl acetate was added and water, and the organic layer separated, washed with brine, dried (Na.sub.2SO.sub.4) andevaporated. The residue was purified by HPLC (10 mm.times.25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (9:1) solvent system. Analytically pure sulfone 12 (22.8 mg, 76%) was collected at Rv 24 mL.
12: .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.262 (3H, s, 7a-H.sub.3), 0.552 (6H, q, J=7.9 Hz, 3.times.SiCH.sub.2), 0.852 (3H, d, J=6.2 Hz, CH.sub.3--CH), 0.935 (9H, t, J=7.9 Hz, 3.times.SiCH.sub.2CH.sub.3), 1.173 [6H, s, C(CH.sub.3).sub.2],2.55 (1H, br d, J=13 Hz, 5.beta.-H), 4.21 (1H, dd, J=14.2, 6.9 Hz, one of CH.sub.2S), 4.43 (1H, dd, J=14.2, 8.9 Hz, one of CH.sub.2S), 5.02 (1H, t, J=7.8 Hz, .dbd.CH), 7.61 (2H, m, Ar--H), 8.00 and 8.22 (1H and 1H, each d, J=8.0 Hz, Ar--H).
1.alpha.,25-Dihydroxy- and 3.beta.,25-dihydroxy-19-norvitamin D.sub.3 analogs (13 and 14). To a solution of sulfone 12 (20.7 mg, 34 .mu.mol) in dry THF (150 .mu.L) was added LiHMDS (1 M in THF, 32 .mu.L, 32 .mu.mol) at -78.degree. C. underargon. The solution turned deep red. The mixture was stirred at -78.degree. C. for 1 h and a solution of the ketone 11 (2.0 mg, 7.1 .mu.mol) in THF (100 .mu.L) was added. The stirring was continued at -78.degree. C. for 2 h, and the reaction mixturewas allowed to warm slowly (for 4 h) to 0.degree. C. After stirring for an additional 30 min at 0.degree. C. it was poured into saturated NH.sub.4Cl and extracted with ether. The extract was washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The yellow oily residue was dissolved in hexane and applied on a silica Sep-Pak. Elution with hexane/ethyl acetate (99.5:0.5, 10 mL and 99:1, 10 mL) and removal of solvents provided the oily residue containing silylated 19-norvitamins (ca. 0.5 mg). The residue was dissolved in dry THF (200 .mu.L) containing Et.sub.3N (3 .mu.L) and treated with tetrabutylammonium fluoride (1.0 M in THF, 20 .mu.l, 20 .mu.mol). The mixture was stirred under argon at room temperature for 17 h, poured into brine, andextracted with ethyl acetate. Organic extract was washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The residue was purified by HPLC (10 mm.times.25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1) solvent system. Isomeric19-norvitamins 13 and 14 (0.2 mg, 6%) were collected at Rv 35 mL and Rv 37 mL. Final purification and separation of both isomers was achieved by reversed-phase HPLC (6.2 mm.times.25 cm Zorbax-ODS column, 2 mL/min) using methanol/water (9:1) solventsystem. 1.alpha.,25-dihydroxyvitamin D analog 13 (120 .mu.g) was collected at Rv 22.5 mL and its isomer 14 (72 .mu.g) at Rv 17.5 mL.
13: UV (in EtOH) .lamda..sub.max 242.0, 251.0, 261.5 nm; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.548 (3H, s, 18-H.sub.3), 0.938 (3H, d, J=6.2 Hz, 21-H.sub.3), 1.219 (6H, s, 26- and 27-H.sub.3), 2.69 (1H, dd, J=11.7, 6.3 Hz, 4.alpha.-H), 2.83(1H, br d, J.about.10 Hz, 9.beta.-H), 3.05 (1H, d, J=14.5 Hz, 10.alpha.-H), 3.62 (1H, dt, J=10.5, 3.4 Hz, one of CH.sub.2--CH.sub.2--O), 3.93 (1H, m, w/2=22 Hz, one of CH.sub.2--CH.sub.2--O), 4.30 (1H, m, w/2=25 Hz, 3.alpha.-H), 4.34 (1H, br s,1.beta.-H), 5.82 (1H, narr m, HC.dbd.C--CH.sub.2), 5.83 and 6.47 (1H and 1H, each d, J=11.0 Hz, 7- and 6-H); HRMS (ESI) exact mass calcd for C.sub.29H.sub.46O.sub.3Na (M.sup.++Na) 465.3345, measured 465.3346.
14: UV (in EtOH) .lamda..sub.max 242.5, 251.0, 261.0 nm; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.551 (3H, s, 18-H.sub.3), 0.939 (3H, d, J=6.2 Hz, 21-H.sub.3), 1.220 (6H, s, 26- and 27-H.sub.3), 2.42 and 2.47 (1H and 1H, each d, J=13.5 Hz,4.alpha.- and 4.beta.-H), 2.82 (1H, br d, J=10.3 Hz, 9.beta.-H), 3.24 (1H, dd, J=12.0, 5.6 Hz, 10.beta.-H), 3.64 and 3.96 (1H and 1H, each m, CH.sub.2--CH.sub.2--O), 4.29 (2H, m, 1.beta.-H overlapped with 3.alpha.-H), 5.84 (1H, m, w/2.about.15 Hz,HC.dbd.C--CH.sub.2), 5.93 and 6.33 (1H and 1H, each d, J=10.5 Hz, 7- and 6-H); HRMS (ESI) exact mass calcd for C.sub.29H.sub.46O.sub.3Na (M.sup.++Na) 465.3346, measured 465.3350.
##STR00007##
##STR00008##
##STR00009##
Biological Activity of 1.alpha.,25-Dihydroxy (Analog 13, REV-B) and 3.beta.,25-Dihydroxy (Analog 14, REV-A) 19-NOR Vitamin D.sub.3 Compounds
The introduction of a heterocyclic ring connecting 3.beta.-oxygen and carbon-2 (analog 13, REV-B) or 1.alpha.-oxygen and carbon-2 (analog 14, REV-A) markedly diminished binding to the full length recombinant rat vitamin D receptor, as compared to1.alpha.,25-dihydroxyvitamin D.sub.3. REV-B had little binding activity for VDR, while REV-A was 3 orders of magnitude less active than 1,25-(OH).sub.2D.sub.3 (FIG. 1). Despite poor receptor binding activity in vitro, these compounds in vivo hadsignificant activity on bone. Thus, REV-A and REV-B are highly selective analogs with unique biological activity.
FIG. 4 demonstrates that REV-A and REV-B have considerable bone calcium mobilization activity, as compared to 1,25(OH).sub.2D.sub.3, at the doses tested.
FIG. 4 thus illustrates that REV-A and REV-B may be characterized as having significant calcemic activity. Their preferential activity on bone calcium mobilizing activity allows the in vivo administration of these compounds for the treatment andprophylaxis of metabolic bone diseases. Because of their preferential activity on bone, these compounds would be preferred therapeutic agents for the treatment and prophylaxis of diseases such as osteoporosis, especially low bone turnover osteoporosis,steroid induced osteoporosis, senile osteoporosis or postmenopausal osteoporosis, as well as osteomalacia and renal osteodystrophy.
FIG. 2 illustrates that REV-A and REV-B are considerably less active than 1,25(OH).sub.2D.sub.3 on HL-60 cell differentiation
FIG. 3 illustrates that the compound REV-A has much less transcriptional activity than 1.alpha.,25-dihydroxyvitamin D.sub.3 in bone cells, while compound REV-B is inactive in this assay. It seems possible that REV-A and REV-B may be converted invivo to free acid forms that possess activity in bone.
The activity of REV-A and REV-B on HL-60 differentiation suggests they will be active in suppressing growth of parathyroid glands and in the suppression of the preproparathyroid gene. These analogs having relatively high calcemic activity arealso expected to be useful as a therapy to treat hypoparathyroidism since they are effective to raise blood calcium levels.
Experimental Methods
The compounds of the invention were prepared and studied using the following methods.
Vitamin D Receptor Binding
Test Material
Protein Source
Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3) Codon Plus RIL cells and purified to homogeneity using two different column chromatography systems. The first system was a nickel affinity resin that utilizes theC-terminal histidine tag on this protein. The protein that was eluted from this resin was further purified using ion exchange chromatography (S-Sepharose
Fast Flow). Aliquots of the purified protein were quick frozen in liquid nitrogen and stored at -80.degree. C. until use. For use in binding assays, the protein was diluted in TEDK.sub.50 (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCI)with 0.1% Chaps detergent. The receptor protein and ligand concentration was optimized such that no more than 20% of the added radiolabeled ligand was bound to the receptor.
Study Drugs
Unlabeled ligands were dissolved in ethanol and the concentrations determined using UV spectrophotometry (1,25(OH).sub.2D.sub.3: molar extinction coefficient=18,200 and .lamda..sub.max=265 nm). Radiolabeled ligand (.sup.3H-1,25(OH).sub.2D.sub.3,.about.159 Ci/mmole) was added in ethanol at a final concentration of 1 nM.
Assay Conditions
Radiolabeled and unlabeled ligands were added to 100 mcl of the diluted protein at a final ethanol concentration of .ltoreq.10%, mixed and incubated overnight on ice to reach binding equilibrium. The following day, 100 mcl of hydroxylapatiteslurry (50%) was added to each tube and mixed at 10-minute intervals for 30 minutes. The hydroxylapaptite was collected by centrifugation and then washed three times with Tris-EDTA buffer (50 mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After the final wash, the pellets were transferred to scintillation vials containing 4 ml of Biosafe II scintillation cocktail, mixed and placed in a scintillation counter. Total binding was determined from the tubes containing only radiolabeled ligand.
HL-60 Differentiation
Test Material
Study Drugs
The study drugs were dissolved in ethanol and the concentrations determined using UV spectrophotometry. Serial dilutions were prepared so that a range of drug concentrations could be tested without changing the final concentration of ethanol(.ltoreq.0.2%) present in the cell cultures.
Cells
Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 medium containing 10% fetal bovine serum. The cells were incubated at 37.degree. C. in the presence of 5% CO.sub.2.
Assay Conditions
HL60 cells were plated at 1.2.times.10.sup.5 cells/ml. Eighteen hours after plating, cells in duplicate were treated with drug. Four days later, the cells were harvested and a nitro blue tetrazolium reduction assay was performed (Collins etal., 1979; J. Exp. Med. 149:969-974). The percentage of differentiated cells was determined by counting a total of 200 cells and recording the number that contained intracellular black-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (data not shown).
In vitro Transcription Assay
Transcription activity was measured in ROS 17/2.8 (bone) cells that were stably transfected with a 24-hydroxylase (24Ohase) gene promoter upstream of a luciferase reporter gene (Arbour et al., 1998). Cells were given a range of doses. Sixteenhours after dosing the cells were harvested and luciferase activities were measured using a luminometer. RLU=relative luciferase units
Antagonism was tested by adding a combination of 1,25(OH).sub.2D.sub.3 and the compound in the same well keeping the final ethanol concentration the same.
Bone Calcium Mobilization
1st In Vivo Study
Female, CD-1 mice (6 weeks old) were purchased from Harlan Sprague-Dawley. Upon arrival, the animals were placed in a room with filtered lighting and fed a purified, D-deficient diet (Suda et al 1970 J. Nutrition) for 17 weeks. After this time,the mice were assigned to 4 groups (n=3/group), and given a single, intraperitoneal injection of vehicle (5% ethanol:95% propylene glycol), 1,25(OH).sub.2D.sub.3, REV-A or REV-B. Blood was collected prior to dose administration and multiple timesthereafter. Serum calcium was measured by diluting with 0.1% lanthum chloride and reading the absorbance using an atomic absorption spectrometer. The change in serum calcium from pre-dose values (baseline) is reported.
2nd In Vivo Study
6-7 week old female CD-1 mice were purchased from Harlan (Indianapolis, Ind.). The animals were group housed and fed a purified diet containing 0.47% calcium. (Suda et al 1970 J. Nutrition). After a 5-7 day acclimation period, the animals wereassigned to treatment groups (n=5-6/group) and given a single dose of the designated analogues by intraperitoneal injection. Blood was collected for serum calcium concentration analyses immediately prior to dose administration and 72 hours followingdose delivery. Serum calcium were analyzed as described above.
3rd In Vivo Study
6-7 week old female CD-1 mice were purchased from Harlan (Indianapolis, Ind.). The animals were group housed and fed a purified diet containing 0.47% calcium. (Suda et al 1970 J. Nutrition). After a 5-7 day acclimation period, the animals wereassigned to treatment groups (n=5/group) and given a single dose of the designated analogues by intraperitoneal injection or oral gavage. Blood was collected for serum calcium concentration analyses at various timepoints following dose delivery. Serumcalcium was analyzed as described above.
Statistical Analysis
In vivo data were analyzed by one-way ANOVA followed by pairwise comparisons when significant overall differences were detected. Post-hoc analyses includes, Tukey's, Scheffe's, and Fisher's LSD tests. Only differences (p<0.05) that werepresent in two out of the three post-hoc tests were considered significant.
Interpretation of Data
VDR binding. HL60 cell differentiation, and transcription activity. REV-A (K.sub.i=3.0.times.10.sup.-8M) and REV-B (K.sub.i=>10.sup.-5M) have much lower ability than the natural hormone 1.alpha.,25-dihydroxyvitamin D.sub.3(K.sub.i=5.0.times.10.sup.-11M) in their ability to compete with [.sup.3H]-1,25(OH).sub.2D.sub.3 for binding to the full-length recombinant rat vitamin D receptor (FIG. 1). Among the synthesized derivatives of 19-nor-1.alpha.,25-(OH).sub.2D.sub.3 REV-Band REV-A, possessing an additional dihydropyrane ring, only the latter has significant binding affinity to the vitamin D receptor albeit decreased more than five hundred times compared to 1,25(OH).sub.2D.sub.3 (FIG. 1). REV-A(EC.sub.50=1.0.times.10.sup.-7M) and REV-B (EC.sub.50=1.0.times.10.sup.-6M) are also considerably lower in their ability to promote HL60 differentiation as compared to 1.alpha.,25-dihydroxyvitamin D.sub.3 (EC.sub.50=2.0.times.10.sup.-9M) (See FIG. 2). Studies on the ability of the vitamins REV-B and REV-A to induce differentiation of human promyelocyte HL-60 cells into monocytes show that their potency is decreased by three and two orders of magnitude, respectively, in comparison with the naturalhormone (FIG. 2). Also, compound REV-A (EC.sub.50=3.0.times.10.sup.-8M) has much lower transcriptional activity in bone cells than 1.alpha.,25-dihydroxyvitamin D.sub.3 (EC.sub.50=2.0.times.10.sup.-10M) while REV-B appears to have essentially notranscriptional activity. (See FIG. 3). Thus, REV-A has weak transcriptional activity, indicated in the 24-hydroxylase (CYP-24) promoter driving luciferase reporter gene system, whereas REV-B has been found completely inactive in this regard (FIG. 3).
Calcium mobilization from bone in vitamin D-deficient animals. Using vitamin D-deficient mice on a low calcium diet (0.02%), the activities of REV-A, REV-B and 1,25(OH).sub.2D.sub.3 in bone were tested. As expected, the native hormone(1,25(OH).sub.2D.sub.3) increased serum calcium levels at the dosage tested (FIG. 4).
Taking into account the above described in vitro results, very low calcemic activity or even complete lack of in vivo activity might be expected for the synthesized analogs REV-B and REV-A. However, studies conducted with these compounds in vivoin D-deficient mice in doses exceeding thirty times that of the natural hormone, show that these compounds have a similar calcemic response after 24 h as calcitriol and isomer REV-B was markedly more active 48 hours after the dose was administered (FIG.4). A second study conducted in D-sufficient CD-1 mice again showed the remarkable in vivo activity of REV-B to raise blood calcium levels (FIG. 5). However, no significant increase in serum calcium was detected with REV-A, which is consistent with theD-deficient mouse study in that the apparent increase in serum calcium after administration of REV-A did not turn out to be statistically significant given the variation and the few animals per group (n=3). Interestingly, the analog that had the leastamount of activity in vitro (REV-B-.alpha.-chair form), has the most in vivo activity and furthermore, its potency in vivo is similar to that of the native hormone. The last experiment conducted with REV-B is shown in FIG. 6. This study was againperformed in D-sufficient CD-1 mice. Two different routes of administration (oral gavage vs. intraperitoneal injection) were studied. Both routes of administration are effective at causing an increase in serum calcium; however, intraperitonealinjection is more effective than oral gavage. This observation is similar to that of the native hormone. It should also be noted that like in the D-deficient animals, there is a significant delay compared to 1,25(OH).sub.2D.sub.3 for an observedincrease in serum calcium to occur when the animals are given REV-B. It can be suggested that in the living organism both analogs might undergo some metabolic changes, possible cleavage of dihydropyrane rings, resulting in one compound with no or verylittle biological activity and the other with pronounced in vivo activity that takes longer to manifest when compared to the native hormone. If ring cleavage is the metabolic conversion taking place, it is not hard to understand why REV-B is much moreactive than REV-A in vivo compared to the in vitro situation as REV-A would not have a 1-hydroxy group which is known to be very important for VDR binding. Further studies are required to prove that ring opening is the explanation for the disparateresults obtained in vivo versus in vitro.
Discussion
Conformational equilibrium of the cyclohexane ring A of vitamin D compounds and its influence on biological activity has been studied for more than three decades. In 1974, Okamura hypothesized that the .beta.-chair form--, which possesses anequatorial 1.alpha.-hydroxyl, is responsible for the biological activities of vitamin D analogs. Our early studies on 1.alpha.,25-dihydroxy-10,19-dihydrovitamin D.sub.3 seemed to contradict this suggestion. Then, the results of biological testing andconformational analysis of 2-methyl substituted analogs of the hormone, synthesized by Japanese scientists, and their 19-nor-counterparts obtained in our laboratory, prompted us to suggest that an axial orientation of 1.alpha.-OH might be of crucialimportance for exertion of calcemic activity. It was found that 2.alpha.-alkylated vitamins, characterized by strong bias (above 90%) toward conformers with the axial hydroxyl at C-1, are much more biologically potent then the respective 2p3-isomersexisting in solution primarily in the opposite .beta.-chair form. Afterward, however, the Moras group reported the crystal structure of the hVDR ligand binding domain (LBD) complexed with calcitriol and several other ligands characterized by anunnatural configuration at C-20. All these results clearly indicated that the receptor binds (at least in the crystalline state) vitamin D compounds having their A-rings in the .beta.-chair conformation. Even more convincing was the recent report fromour laboratory, in which Vanhooke described the crystal structure of the rat VDR LBD complexed with a 2.alpha.-methyl-substituted vitamin D.sub.3 analog, possessing highly elevated biopotency in both intestine and bone. The study shows that this vitaminD compound also adopts the .beta.-chair A-ring conformation in its crystalline complex with VDR. However, in all these cases, interconversion between the ligand's A-ring chair conformers was not prohibited, and therefore, some doubts could arise whichform of the ligand-VDR complex actually exists in the real physiological environment. Thus, it was tempting to synthesize and biologically evaluate a vitamin D compound possessing an axially oriented hydroxyl group at C-1 and unable to change its A-ringconformation. The 1.alpha.,25-dihydroxyvitamin D.sub.3 analog REV-B, described in the present work, fulfills these requirements. It was established that such vitamin D does not bind to the VDR and lacks activity in cellular differentiation and ininducing transcription of a vitamin D-responsive gene. Notably, its isomer REV-A possessing free 3.beta.- and 25-hydroxyl groups, but characterized by a "frozen" A-ring .beta.-chair conformation, was found to be ca. 560 times less potent in binding tothe receptor than the hormone. Such binding ability could be expected for a 25-OH-D.sub.3 derivative in which the 1.alpha.-oxygen function cannot act as a hydrogen donor and create the hydrogen bonds with the amino acids from the LBD. The biologicalresults obtained in vitro on the synthesized analogs REV-B and REV-A clearly confirm that the A-ring .beta.-chair conformation and, consequently, equatorial orientation for the 1.alpha.-OH is necessary for the vitamin D compound to ensure its bindingwith VDR and exertion of biological activity. Biological evaluation of the test vitamins in vivo does not generate results consistent with those obtained in vitro, most likely due to metabolic transformation of both compounds occurring in the livingorganisms.
Conclusions
The conformations of the A-rings of vitamins REV-B and REV-A, as well as the structures of intermediate compounds used for the construction of their A-ring parts, were established by NMR spectroscopic methods. Analysis of the observed vicinalproton coupling constants proved that in the synthesized vitamin D analogs REV-B and REV-A, possessing an additional dihydropyrane ring, their A-rings could only exist in a single confirmation, .alpha.- and .beta.-chair, respectively. Biological invitro testing of the analogs REV-B and REV-A allowed us to conclude that the presence of an equatorially oriented free hydroxyl at C-1 is necessary for binding to the vitamin D receptor. Thus, only the vitamin D that can assume a .beta.-chair A-ringconformation can be accepted by the VDR further inducing conformational changes crucial for the ligand-receptor activation.
These results illustrate that REV-A and REV-B are excellent candidates for numerous human therapies as described herein, and that they may be particularly useful in a number of circumstances such as suppression of secondary hyperparathyroidism ofrenal osteodystrophy. The fact that REV-A and REV-B in vivo have impressive activity on bone suggests that they would be useful in treating metabolic bone diseases, especially renal osteodystrophy, osteoporosis, osteopenia, vitamin D-resistant rickets,and osteomalacia.
The compounds of the invention of formula I and II are also useful in preventing or treating obesity, inhibiting adipocyte differentiation, inhibiting SCD-1 gene transcription, and/or reducing body fat in animal subjects. Therefore, in someembodiments, a method of preventing or treating obesity, inhibiting adipocyte differentiation, inhibiting SCD-1 gene transcription, and/or reducing body fat in an animal subject includes administering to the animal subject, an effective amount of one ormore of the compounds or a pharmaceutical composition that includes one or more of the compounds of formula I and/or II. Administration of the compound or the pharmaceutical compositions to the subject inhibits adipocyte differentiation, inhibits genetranscription, and/or reduces body fat in the animal subject. The animal may be a human, a domestic animal such as a dog or a cat, or an agricultural animal, especially those that provide meat for human consumption, such as fowl like chickens, turkeys,pheasant or quail, as well as bovine, ovine, caprine, or porcine animals.
For prevention and/or treatment purposes, the compounds of this invention defined by formula I and II may be formulated for pharmaceutical applications as a solution in innocuous solvents, or as an emulsion, suspension or dispersion in suitablesolvents or carriers, or as pills, tablets or capsules, together with solid carriers, according to conventional methods known in the art. Any such formulations may also contain other pharmaceutically-acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying or taste-modifying agents.
The compounds of formula I and II may be administered orally, topically, parenterally, rectally, nasally, sublingually, or transdermally. The compound is advantageously administered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentary canal, or in the form of creams, ointments, patches, or similar vehicles suitable for transdermal applications. A dose of from 0.01 .mu.g to 10 mg per day of the compounds I or II,preferably from about 0.1 .mu.g to about 1 mg per day, is appropriate for prevention and/or treatment purposes, such dose being adjusted according to the disease to be treated, its severity and the response of the subject as is well understood in theart. Since the compounds exhibit specificity of action, each may be suitably administered alone, or together with graded doses of another active vitamin D compound--e.g. 1.alpha.-hydroxyvitamin D.sub.2 or D.sub.3, or 1.alpha.,25-dihydroxyvitaminD.sub.3--in situations where different degrees of bone mineral mobilization and calcium transport stimulation is found to be advantageous.
Compositions for use in the above-mentioned treatments comprise an effective amount of the compounds I or II as defined by the above formula I and II as the active ingredient, and a suitable carrier. An effective amount of such compound for usein accordance with this invention is from about 0.01 .mu.g to about 10 mg per gm of composition, preferably from about 0.1 .mu.g to about 1 mg per gram of composition, and may be administered topically, transdermally, orally, rectally, nasally,sublingually or parenterally in dosages of from about 0.01 .mu.g/day to about 10 mg/day, and preferably from about 0.1 .mu.g/day to about 1 mg/day.
The compounds I and II may be formulated as creams, lotions, ointments, topical patches, pills, capsules or tablets, suppositories, aerosols, or in liquid form as solutions, emulsions, dispersions, or suspensions in pharmaceutically innocuous andacceptable solvent or oils, and such preparations may contain in addition other pharmaceutically innocuous or beneficial components, such as stabilizers, antioxidants, emulsifiers, coloring agents, binders or taste-modifying agents.
The compounds I and II may be advantageously administered in amounts sufficient to effect the differentiation of promyelocytes to normal macrophages. Dosages as described above are suitable, it being understood that the amounts given are to beadjusted in accordance with the severity of the disease, and the condition and response of the subject as is well understood in the art.
The formulations of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of beingcompatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder orgranules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.
Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and carrier such as cocoa butter, or in the form of an enema.
Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient.
Formulations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such asdrops; or as sprays.
For nasal administration, inhalation of powder, self-propelling or spray formulations, dispensed with a spray can, a nebulizer or an atomizer can be used. The formulations, when dispensed, preferably have a particle size in the range of 10 to100.mu..
The formulations may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. By the term "dosage unit" is meant a unitary, i.e. a single dose which is capable of beingadministered to a patient as a physically and chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.
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